Figure 2

AP-2 isoform and conditional ΔAP-2γ expression in N202.1A cells. Verification of ΔAP-2γ function using BMP-4 luciferase promoter assays. (A) Western-blot using antibodies detecting the different AP-2 isoforms in N202.1A cells. As positive control (Pos) for the antibodies, we used protein lysate from HeLa cells for AP-2α (HeLa), N2A cells for AP-2δ (N2A), human Keratinocytes for AP-2ε (hum Ker) and in vitro translated protein for AP-2β (iv). (B) Northern blot analysis of ΔAP-2γ expression in the stable N202.1A clones, uninduced or induced with doxycycline (2 μg/ml). Fifteen micrograms of total RNA were resolved on a formaldehyde gel, transferred to a Nylon membrane and hybridized with a P-32-labelled AP-2γ cDNA probe. GAPDH probe was used to monitor the amounts of RNA. (C) N202.1A cells were induced with doxycycline (2 μg/ml) for 96h and subjected to western blot analysis. Δ#7 and Δ#15 show strong transgene expression in a doxycyline dependent-manner (32 kDa), which is not detectable in Co#5 and Co#11. Of note, the antibody also detects endogeneous AP-2γ (50 kDa) which is not affected by doxycycline. (D) HCT116 cells were transfected with expression constructs for AP-2α and ΔAP-2γ. Co-IP experiment using antibody to AP-2γ for IP and antibody to AP-2α to detect heterodimerization between ΔAP-2γ and AP-2α. - no Antibody; + IP using AP-2γ Antibody; input control. (E) For BMP-4-promoter luciferase assays N202.1A cells were induced with doxycycline for 96 h and transfected with BMP-4 luciferase. 48 h after transfection luciferase activity was quantified. For internal normalization of transfection efficiency a CMV-driven renilla luciferase was used.